Screening of basidiomycetes for lignin peroxidase genes using a DNA probe

135

. In all cases, ⅐ OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H 2 O 2 levels and Fe 3؉ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher ⅐ OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between ⅐ OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of ⅐ OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.

Section: Methodsmentioning

confidence: 99%

Induction of Extracellular Hydroxyl Radical Production by White-Rot Fungi through Quinone Redox Cycling

Gómez-Toribio

García-Martín

Martı́nez

et al. 2009

135

“…We evaluated in vitro decay of Eucalyptus globulus wood (obtained from the ENCE mill in Pontevedra, Spain) under solid-state fermentation conditions. Two grams (dry weight) of small chips (1 to 2 by 10 to 20 mm) and 4 ml of water were placed in 100-ml Erlenmeyer flasks, sterilized for 20 min at 120°C, and then inoculated with pellets from a fungal culture; the culture was grown in glucose-peptone medium (36) and had been washed and resuspended in 1.5 ml of sterile water (the dry weight of inoculum was 0.5 mg per g of wood). The cotton-plugged flasks were covered with aluminum foil to maintain the humidity and incubated (static) at 28°C for 7 weeks.…”

Section: Methodsmentioning

confidence: 99%

Production of New Unsaturated Lipids during Wood Decay by Ligninolytic Basidiomycetes

Gutiérrez

Río

Martinez-Inigo

et al. 2002

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.Fungi produce a variety of lipids including fatty acids in free or esterified form, e.g., glycerides, phospholipids, glycolipids, sterol esters, sphingolipids, or simple esters, as well as other lipids, e.g., free sterols, sterol glycosides, and hydrocarbons (46,54,56). Vegetative mycelium (from pure cultures) and fruit bodies of basidiomycetes differ in both lipid content and fatty acid profiles. Linoleic (18:2 9c,12c ), oleic (18:1 9c ), and palmitic (16:0) acids are the main fatty acids in most cases. Linoleic acid is very abundant in fruit bodies and may account for as much as 70 to 80% of the lipids present (1,42,48,49). Other saturated and unsaturated fatty acids are present in basidiomycetes at lower levels. As reported for other filamentous fungi, unsaturated fatty acids could help basidiomycetes adapt to low growth temperatures (11,12). These fatty acids also may have a role in lignin degradation.Lipid peroxidation reactions could be a part of the lignin degradation process by white rot basidiomycetes. When unsaturated fatty acids are oxidized in the presence of manganese peroxidase (MnP), a ligninolytic peroxidase that oxidizes Mn 2ϩ to Mn 3ϩ , lipid hydroperoxides and free radicals are formed. This reaction is considered to be sufficient for lignin degradation by some ligninolytic fungi, e.g., Ceriporiopsis (syn. Poria) subvermispora, that lack lignin peroxidase (LiP) (4, 31-33). LiP, which has a high redox potential that enables direct oxidation of nonphenolic lignin units, was described in the white rot fungus Phanerochaete chrysosporium together with MnP (37). However, LiP has not been detected in some efficient lignin-degrading...

“…They were first described in Polystictus versicolor (5). AAO activity was later detected in different fungi, including Pleurotus species (6)(7)(8)(9), Fusarium solani (10), Rigidoporus microporus (synonym, Fomes lignosus) (11), Bjerkandera adusta (12,13), and Botrytis cinerea (14). The simultaneous production of AAO and lignin peroxidase (LiP) in B. adusta (15) and of AAO and versatile peroxidase in Pleurotus cultures (16) supports AAO involvement in lignin degradation.…”

mentioning

confidence: 99%

Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass

Mathieu

Piumi

Valli

et al. 2016

Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger. Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (P. cinnabarinus GDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii. Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol: quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus.A uxiliary activities (AA) are a widespread group of catalytic modules involved in plant cell wall degradation. They were first introduced in 2013 into the carbohydrate-active enzymes (CAZy [www.cazy.org]) database (1). This AA class encompasses and extends an earlier classification dedicated to fungal ligninolytic enzymes and comprises 10 families, some of which have subfamilies (2). The AA family group enzymes possess the potential ability to help the original carbohydrate-active enzymes gain access to the carbohydrates embedded in the plant cell wall. Lignin, the main noncarbohydrate structural component of plant cell walls, consists of an intricate network of aromatic compounds forming a strong, hydrophobic, insoluble barrier closing access to the carbohydrates. Microorganisms have therefore set up different strategies to either modify or degrade lignin as a first step to gain access to the carbohydrate moiety and convert it into an energy source (3).In this new AA classification, family AA3 belongs to the glucose-methanol-choline (GMC) oxidoreductase family first defined by Cavener (4). AA3 enzymes are flavoproteins containing a flavin adenine dinucleotide (FAD)-binding domai...